Long term survival and growth within host macrophages is a unique bacterial lifestyle shared by many important human pathogens. The mechanisms of intracellular persistence are, for the most part, unknown. Brucella abortus, a facultative intracellular pathogen that causes chronic infection in humans, can be used to study intracellular persistence because it lends itself to genetic analysis. The goal of the proposed study is the identification and characterization of B. abortus genes involved in survival and persistence in host cells. To this end, we will generate a bank of 1000 signature-tagged transposon mutants of B. abortus and screen the mutants for defects in host persistence. Mixed pools of B. abortus mutants will be used to infect mice and after 8 weeks, bacteria will be recovered from each mouse. The composition of the mutant pool recovered from the mouse after 8 weeks will be compared with that of the pool originally used to infect the mouse. Since each mutant carries a unique oligonucleotide tag, it will be possible to identify which mutants failed to establish or maintain infection in the mouse by detection of the tag. Mutants unable to persist in the mouse will be characterized by sequence analysis of the inactivated gene, chromosomal mapping of the transposon insertion and assays for survival and trafficking in host macrophages. This approach promises to uncover new mechanisms of establishing chronic intracellular infection, and to greatly improve our current understanding of B. abortus infection. In turn, this knowledge will facilitate research with other bacterial intracellular pathogens which are more difficult to analyze genetically.